1. Volume 24, Issue 6, Pages 1573-1584 (August 2018)
Fan-Shaped Body Neurons in the Drosophila Brain Regulate Both Innate and Conditioned Nociceptive Avoidance 
Wantong Hu, Yiqing Peng, Jiameng Sun, Fang Zhang, Xuchen Zhang, Lianzhang Wang, Qian Li, Yi Zhong 
Cell Reports 
Volume 24, Issue 6, Pages (August 2018)
DOI: /j.celrep
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2. Cell Reports 2018 24, 1573-1584DOI: (10.1016/j.celrep.2018.07.028)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1 Strong Response of the FB to ES
(A) Illustration of in vivo two-photon imaging. Fly genotype is shown on the right panel. ES (60 V; 0.1 Hz; 1 min) was delivered to the legs of a single fly through conductive gel.
(B) Representative calcium responses of a single fly during 1 min ES (left panel). Responses from several regions were recorded, including the MB (calyx, KC, peduncle, vertical lobes, and horizontal lobes), FB, and EB. KC stands for the cell bodies of Kenyon cells. The FB (red line) had the strongest response to ES.
(C) Shock-induced calcium responses of all flies. Gray lines, individual data (n = 6–7); red lines, mean value.
(D) Statistics of peak Df/f values of the shock-evoked responses. The peak value of the FB was significantly higher than in other regions. The error bars represent mean ± SEM (n = 6–7). ∗p < 0.05.
(E) Spectrum analysis was applied to data. A power of 0.1 Hz represented the relative calcium response intensity to ES.
(F) Statistics for 0.1 Hz power for all regions. The 0.1-Hz power value of the FB was significantly higher than in other regions. The error bars represent mean ± SEM (n = 6–7). ∗p < 0.05.
See also Figure S1.
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4. Figure 2 Different ES-Induced Responses in the Different FB Layers
(A) Expression pattern and schematic graph of four Gal4 drivers in the FB region (R49H02, R89E07, R38E07, and C205). Green, neurons labeled by each Gal4 line (upper left panel); magenta, neuropils labeled by nc82 antibody (upper right panel). Merged (lower left panel) is shown. Schematic graph shows specific layers labeled by each Gal4 line for the 9 layers (lower right panel). The scale bar represents 22 μm.
(B) Representative calcium responses of a single fly during 1 min of 60 V ES. Different layers are shown separately, with layer numbers marked on the left.
(C) Statistics for 0.1 Hz power for all flies, with layers separated. The ventral and middle layers (L1, L2, L4, and L5) showed significantly higher values than the dorsal layers (L6, L8, and L9). The error bars represent mean ± SEM (n = 6–14). ∗p < 0.05.
See also Figures S1, S2, S3, S4, and S5.
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5. Figure 3
Requirements of FB Neurons in Avoiding Acute Nociceptive Stimuli
(A) Blocking FB neurons impaired the ability of flies to avoid ES stimuli. An illustration of the shock-avoidance paradigm is shown on the left. Flies were allowed to choose freely between two arms of the T-maze. ES (30 V) was delivered to one arm alternatively. Two blocking tools were used: shibirets (middle panel) and Kir 2.1 (right panel). Blocking neurons of ES-responsive Gal4 lines (R49H02, R89E07, and R38E07; dark bars), but not ES-non-responsive Gal4 line (C205; dark bar), led to a decrease in the shock-avoidance index compared with the control groups (white bars). The error bars represent mean ± SEM (n = 6–14). ∗p < Genotypes are described at the bottom of each graph.
(B) Illustration of the optogenetic activation paradigm (left). Flies were allowed to choose freely between two arms of the T-maze. Orange light was delivered to one arm alternatively to activate ReaChR. Activating neurons of the ES-responsive Gal4 lines (R49H02, R89E07, and R38E07; dark bars) resulted in a higher avoidance index than in controls (white bars). The ES-non-responsive Gal4 line (C205; dark bar) showed no significant changes. The error bars represent mean ± SEM (n = 6–17). ∗p < Genotypes are described at the bottom of each graph.
(C) Illustration of noxious heat-avoidance test (left). Flies were gently transferred onto a 48°C water bath for 4 min. The flies that could not avoid the noxious became immobile and counted toward the avoidance index. Blocking neurons of the ES-responsive Gal4 lines (R49H02, R89E07, and R38E07; dark bars), but not the ES-non-responsive Gal4 line (C205; dark bar), led to a decrease in the heat-avoidance index compared with the control groups (white bars). The error bars represent mean ± SEM (n = 12–20). ∗p < 0.05.
See also Figures S4, S5, S6, and S7.
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6. Figure 4
Activation of FB Neurons Could Serve as Harmful Stimuli in Conditioning
(A) Illustration of artificial activation-reinforced conditioning. Neurons in flies carrying TrpA1 were activated at 26°C. In the training stage, an odor was presented to flies with or without conditioning with FB neurons activation. In the testing stage, flies were allowed to choose between the two odors that appeared during training.
(B) Performance index of artificial activation-reinforced conditioning. Artificial activation of FB neurons (dark bars) labeled by R49H02, R89E07, and R38E07, but not C205, displayed significantly higher scores than controls (white bars). The error bars represent mean ± SEM (n = 6–13); ∗p < 0.05.
See also Figure S5.
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7. Figure 5 Involvement of FB Neurons in Avoiding Conditioned ES Stimuli
(A) Illustration of Pavlov’s canonical olfactory conditioning in Drosophila. In the training stage, ES was paired with one odor. In testing stage, flies were allowed to choose between the two odors that appeared during training.
(B) Schematic of experiments. Flies were trained and tested at ether a permissive 23°C or a restrictive 30°C. The temperature was raised 15 min prior to training or testing. For the group, training and testing both took place at 23°C (left). For the group, training took place at 30°C and testing took place at 23°C (middle). For the group, training took place at 23°C and testing took place at 30°C (right).
(C) Blocking FB neurons impaired the flies’ ability to avoid learned harmful stimuli. Compared to the group (white bars) and the group (gray bars), the group (black bars) showed a significant decrease, except for R38E07. The error bars represent mean ± SEM (n = 8–11); ∗p < Genotypes are described at the bottom.
(D) Illustration of appetitive olfactory conditioning in Drosophila. In the training stage, sugar was paired with one odor. In the testing stage, flies were allowed to choose between the two odors that appeared during training.
(E) Blocking FB neurons did not impair the flies’ learned attraction to sugar. Compared to the group (white bars) and the group (gray bars), the group (black bars) showed no significant decrease. The error bars represent mean ± SEM (n = 9–11). Genotypes are described at the bottom.
See also Figure S6.
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8. Figure 6 Model of FB Neurons Involved in Different Avoidance
Among large-field neurons detected in this study, L1, L2, L4, and L5 showed high ES response (left panel, red) and L6, L8, and L9 showed low ES response (left panel, blue). As to innate avoidance, L1, L2, L4, and L5 were involved (top right panel). As to conditioned avoidance, L2 and L6 were involved (bottom right panel). L1 and L4 neurons participate in innate avoidance; however, their role in conditioned avoidance is difficult to determine on the basis of the data available.
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